Circulating antigens of dirofilaria immitis, monoclonal antibodies specific therefor and methods of preparing such antibodies and detecting such antigens
First Claim
1. Circulating parasite antigens of Dirofilaria immitis essentially purified and isolated from Dirofilaria immitis adult worms or infected dog serum treated with trichloroacetic acid and heat, said antigens characterized as follows:
- (a) the antigens being present in Dirofilaria immitis worms and in the serum of animals infected with Dirofilaria immitis;
(b) being high molecular weight parasite antigens in infected dog sera as demonstrated by the immunoblot method with polyclonal and monoclonal antibodies;
(c) not being destroyed by trichloroacetic acid extraction or by perchloric acid extraction;
(d) not being destroyed by heat treatment at approximately 100°
C. for 30 minutes;
(e) having isoelectric points of under 4 as determined by a combination of ion exchange chromatography and rocket-line immunoelectrophoresis;
(f) migrating towards the anode at pH 8.6 in agarose gel (10 V/cm) with migration distances relative to albumin of 1.1 and about 1.0 as determined by crossed immunoelectrophoresis;
(g) forming lines of identity in rocket-line immunoelectrophoresis with soluble acid and heat-stable antigens extracted from adult D. immitis worms;
(h) being a subset of antigens produced in vitro by adult female D. immitis worms;
(i) having a phosphocholine determinant; and
(j) having determinants whose immunoreactivity is ablated by treatment thereof with sodium metaperiodate.
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Accused Products
Abstract
Novel hybrid cell lines are disclosed for providing monoclonal antibodies to circulating parasite antigens present in the blood of animals infected with the nematode parasite Dirofilaria immitis. Such circulating parasite antigens are also characterized and identified. Further disclosed are methods employed to produce and select such hybrid cell lines and diagnostically useful assay methods utilizing the monoclonal antibodies in the detection of such specific circulating parasite antigens in blood or bodily fluids.
45 Citations
14 Claims
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1. Circulating parasite antigens of Dirofilaria immitis essentially purified and isolated from Dirofilaria immitis adult worms or infected dog serum treated with trichloroacetic acid and heat, said antigens characterized as follows:
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(a) the antigens being present in Dirofilaria immitis worms and in the serum of animals infected with Dirofilaria immitis; (b) being high molecular weight parasite antigens in infected dog sera as demonstrated by the immunoblot method with polyclonal and monoclonal antibodies; (c) not being destroyed by trichloroacetic acid extraction or by perchloric acid extraction; (d) not being destroyed by heat treatment at approximately 100°
C. for 30 minutes;(e) having isoelectric points of under 4 as determined by a combination of ion exchange chromatography and rocket-line immunoelectrophoresis; (f) migrating towards the anode at pH 8.6 in agarose gel (10 V/cm) with migration distances relative to albumin of 1.1 and about 1.0 as determined by crossed immunoelectrophoresis; (g) forming lines of identity in rocket-line immunoelectrophoresis with soluble acid and heat-stable antigens extracted from adult D. immitis worms; (h) being a subset of antigens produced in vitro by adult female D. immitis worms; (i) having a phosphocholine determinant; and (j) having determinants whose immunoreactivity is ablated by treatment thereof with sodium metaperiodate. - View Dependent Claims (2, 3, 4, 5, 6, 13)
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- 7. An assay method for determining the presence of circulating parasite antigens of Dirofilaria immitis in the serum of plasma of D. immitis-infected dogs which comprises combining a sample of serum or plasma from a dog infected with Dirofilaria immitis and to which no parasite antigen or extract derived from D. immitis is added with a first antibody bound to the surface of a solid support, adding thereto a conjugate composed of a second antibody and an enzyme and thereafter adding an enzyme substrate to indicate the presence of absence of said circulating parasite antigens in said sample.
- 11. An assay method for determining the presence of circulating parasite antigens of Dirofilaria immitis in the serum of plasma of D. immitis-infected dogs which comprises combining a sample of serum or plasma from a dog infected with Dirofilaria immitis and to which no parasite antigen or extract derived from D. immitis is added with a first monoclonal antibody which specifically binds to determinants present on said circulating parasite antigens, and detecting the presence of said antigens by means of a label on said first antibody or by means of a label on a second antibody added to said first monoclonal antibody, said second antibody specifically binding to said circulating parasite antigens.
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14. An assay method for determining the presence of circulating parasite antigens of Dirofilaria immitis in a sample of serum or plasma of D. immitis-infected dogs to which no parasite antigen or extract derived from D. immitis is added which comprises the steps of:
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(a) attaching a first antibody to a solid support, said first antibody being a polyclonal antibody which binds to antigenic determinants of said circulating parasite antigens of Dirofilaria immitis; (b) combining said first antibody with said sample of serum or plasma from a dog infected with Dirofilaria immitis and a conjugate composed of an enzyme and a monoclonal antibody which binds to antigenic determinants of said circulating parasite antigens of Dirofilaria immitis whereby a polyclonal antibody-antigen-conjugated monoclonal antibody sandwich is formed; and (c) thereafter adding an enzyme substrate to said sandwich to colorimetrically indicate the presence or absence of said circulating parasite antigens.
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Specification